ABSTRACT The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol by reducing cytochrome c. Cytochrome b, the catalytic core of bc1, generates superoxide during the oxidation of ubiquinol. Excessive superoxide production is known to accelerate aging and neurodegeneration. Songbirds (oscine, Passeri) exhibit lower production of mitochondrial reactive oxygen species (ROS) and greatly accelerated evolution of cytochrome b, relative to all other modern birds, suggesting adaptive selection for lower generation of ROS. Here, we identified songbird-specific substitutions in modern bird's cytochrome b amino-acid sequences and examined the high-resolution structures of the chicken bc1 complex in an effort to predict the effect of these substitutions on the function of bc1. Many of the songbird-specific substitutions cluster around sites that are critical for the function of bc1. One cluster of substitutions interacts with heme BH. A second cluster of substitutions interacts with residues in the ubiquinone reduction site, Qi. Both groups of substitution may affect the rate of reduction of ubiquinone at the Qi site. Another cluster of cytochrome b substitutions interacts with the hinge region of the Rieske protein that transfers electron from cytochrome b to cytochrome c1. These songbird-specific substitutions appear to be selected to modulate the rate of both ubiquinol oxidation at the Qo site and ubiquinone reduction at the Qi site thereby modulating the rate of superoxide production. These findings are compatible with the hypothesis that cytochrome b evolution in songbirds was driven by selection of substitutions that reduce the rate of superoxide production thereby increasing songbird lifespan and cognitive abilities.